Valorization of Canteen Wastewater Through Optimized Spirulina Platensis Cultivation for Enhanced Carotenoid Production and Nutrient Removal

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please check my comments, attached document Phycology-4007245

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 1

Comment 1: Figure 1 and 2 express same data please decide on one of these data.

Response 1: We thank the reviewer for pointing out this redundancy. We agree that presenting both Optical Density (OD) and Dry Biomass Weight for the same experiment was unnecessary in the main text. To resolve this, we have moved the OD growth curves (formerly Figure 1) to the Supplementary Materials (Figure S1). We have retained the Dry Biomass Weight graph (now Figure 1) in the main manuscript, as dry weight provides a more accurate quantitative measure for calculating biomass productivity.

Comment 2: Figure 3 and 4 same, express same thing. Please explain why data of figure 3 are different from table 1.

Response 2:

• Regarding Redundancy: Similar to the previous comment, we have moved the OD data for the light intensity screening (formerly Figure 3) to the Supplementary Materials (Figure S2) and kept the Dry Biomass Weight graph (now Figure 2) in the main text to avoid repetition.

• Regarding the Data Difference: The reviewer correctly identified a difference between the screening data (Figure 3/Figure S2) and the main experiment data (Table 1). The screening experiments were conducted in 500 mL Erlenmeyer flasks, whereas the main experiment (Table 1) was performed in 2 L photobioreactors. The larger reactor volume provided superior mixing efficiency and gas exchange, resulting in significantly higher biomass accumulation (OD ~1.88) compared to the screening phase (OD ~1.16). We have added an explicit explanation for this observation in Section 3.3 of the revised manuscript to clarify the discrepancy.

Comment 3: Also, are the figure 4, 5 and table 2 data different? Please explain.

Response 3: Yes, the data sources are different. Figure 4 (now Figure 2) presents data from the preliminary screening phase conducted in 500 mL flasks to select the optimal light intensity. In contrast, Figure 5 (now Figure 3) and Table 2 (now Table 1) present data from the main experiment, which was conducted in 2 L bioreactors using the optimized conditions identified in the screening. As noted in Response 2, the scale-up to 2 L vessels improved growth conditions, leading to the higher values observed in Figure 5 and Table 2 compared to Figure 4. We have clarified the experimental setup for each phase in Section 3 to prevent confusion.

Comment 4: Conclusion: It is not possible reach any conclusion without analyze your present results. Check all to be consistent.

Response 4: We appreciate this constructive feedback. We have completely rewritten the Conclusion (Section 5) to ensure it is directly grounded in the results analyzed in the study. The revised conclusion now quantifies the specific key findings, including the biomass productivity (0.071 g L⁻¹ day⁻¹), nutrient removal efficiencies (>90%), and the trade-off between protein and lipid/carotenoid content. We have removed generic statements and ensured that all conclusions are supported by the presented data.

Reviewer 2 Report

Comments and Suggestions for Authors

This article summarizes valorization of canteen wastewater by Spirulina platensis cultivation to obtain high value products such as proteins and carotenoids. Optimization conditions include canteen wastewater percentage and light intensity; and the biomass productivity reached 0.071 g L⁻¹ day⁻¹. The obtained algal biomass contained 54.3% DW proteins and 21.81 mg g⁻¹ DW carotenoids. The study is comprehensive; the work is sound. Given the fact that valorization of wastewater into high value products is a highly recognized area of research, this study will make a significant contribution to the field and will attract the attention of researchers from a wide range of fields.
However, there are several minor issues that prevent the publication of the work in its current form:
1. The obtained algal biomass content under optimized conditions (54.3% DW proteins and 21.81 mg g⁻¹ DW carotenoids) was reported and compared with the literature data for carotenoid content of Spirulina platensis cultivation. However, authors should determine the protein and carotenoid content of the biomass obtained from various CW percentages and control BG11 cultivation medium and compare.

2. All Figures must be drawn with the same format, similar line settings etc. Figure 4, please keep x-axis division at 2 units, similar to Figure 3.
3. Table headings must be re-written (Table 1: Optical Density (OD600) of Spirulina sp. cultivated in optimized canteen (what? Canteen wastewater??)).
4. Table columns must be named precisely (Table 1: 180 µmol what?? Is this light intensity??, 180 µmol m⁻² s⁻¹).
5. Line 355: Table 1 must actually be Table 5.
6. Abbreviation CW is used for canteen wastewater, however its use is inconsistent. For example, at Table 3 and Line 355, Table 1 abbreviation WW is used.

Author Response

Comment 1: The obtained algal biomass content under optimized conditions (54.3% DW proteins and 21.81 mg g⁻¹ DW carotenoids) was reported... However, authors should determine the protein and carotenoid content of the biomass obtained from various CW percentages and control BG11 cultivation medium and compare.

Response 1: We appreciate this valuable suggestion. To address this, we have conducted a comparative biochemical analysis for the BG11 Control alongside the optimized wastewater culture. We have updated Table 4 (formerly Table 2) and Section 3.5 to include the protein, carbohydrate, lipid, and carotenoid content for the control samples.

• Result: The comparison reveals that while the control biomass had slightly higher protein content (61.5% vs. 54.3%), the wastewater-grown biomass exhibited significantly higher lipid content (7.85% vs. 5.90%) and carotenoid yield (21.81 mg g⁻¹ vs. 6.85 mg g⁻¹). This data strongly supports the conclusion that wastewater cultivation enhances the production of high-value secondary metabolites and lipids.

Comment 2: All Figures must be drawn with the same format, similar line settings etc. Figure 4, please keep x-axis division at 2 units, similar to Figure 3.

Response 2: We have revised all figures to ensure a uniform appearance. Specifically, the x-axis for the dry biomass growth curves (now Figure 1 and Figure 2) has been standardized to show 2-day intervals (0, 2, 4, 6...) as requested. Line thickness, marker sizes, and font styles have been harmonized across all figures to improve visual consistency.

Comment 3: Table headings must be re-written (Table 1: Optical Density (OD600) of Spirulina sp. cultivated in optimized canteen (what? Canteen wastewater??)).

Response 3: We apologize for the lack of clarity in the original draft. We have rewritten all table captions to be precise and descriptive. For example, the caption for the main growth experiment now reads: "Table 1. Dry biomass concentration of Spirulina sp. cultivated in optimized Canteen Wastewater (CW) vs BG11."

Comment 4: Table columns must be named precisely (Table 1: 180 µmol what?? Is this light intensity??, 180 µmol m⁻² s⁻¹).

Response 4: We have corrected the column headers in all tables to include the full units of measurement. Headers now explicitly state "180 µmol photons m⁻² s⁻¹" or "(g L⁻¹)" where appropriate to avoid ambiguity.

Comment 5: Line 355: Table 1 must actually be Table 5.

Response 5: We have corrected the table numbering throughout the manuscript. Tables are now numbered sequentially from Table 1 (Growth Results) to Table 5 (Literature Comparison in the Discussion). All in-text citations have been updated to match the correct table numbers.

Comment 6: Abbreviation CW is used for canteen wastewater, however its use is inconsistent. For example, at Table 3 and Line 355, Table 1 abbreviation WW is used.

Response 6: We thank the reviewer for spotting this inconsistency. We have performed a thorough review of the entire text, tables, and figure legends. All instances of "WW" or "wastewater" (when referring to the specific medium) have been standardized to the abbreviation "CW" (Canteen Wastewater) to ensure consistency throughout the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Review for the paper “Valorization of Canteen Wastewater through Optimized Spirulina platensis Cultivation for Enhanced Carotenoid Production and Nutrient Removal” by Charith Akalanka Dodangodage and co-authors submitted to “Phycology”.

The authors of this research paper conducted an analysis to evaluate the viability of utilizing canteen wastewater as a sustainable cultivation medium for the microalgae Spirulina platensis, with the aim of integrating wastewater remediation with the production of valuable biomass. They employed a comparative experimental design, assessing algal growth, nutrient removal efficiency, and the accumulation of high-value carotenoids against a conventional synthetic medium. They found that a diluted formulation of the wastewater supported significantly superior microalgal growth and biomass productivity compared to the standard control medium. The process achieved remarkable efficiency in removing nitrogen and phosphorus pollutants from the wastewater stream. The cultivated biomass was enriched with carotenoids and proteins, demonstrating the successful conversion of waste nutrients into potentially valuable biochemical compounds. The results of this study may have important implications for the development of circular bioeconomy models within institutional settings.

Recommendations.

Introduction

L 36-39. I think it would really help if the authors could give a quick explanation or maybe an example of "qualitative degradation" besides just nutrient pollution. Adding some references here could make it clearer too.

L 40-42. The authors should back up their claim about the rapid growth in wastewater generation with a citation.

L 66-67. I would suggest that the authors share the typical tolerance ranges for Spirulina in wastewater conditions.

Materials and Methods

L 117. I think it would be helpful if the authors briefly mentioned the key criteria they used to pick the optimal 75% dilution based on the screenings, like biomass yield or growth rate.

L 141. I am curious why the authors chose the stationary phase instead of the exponential phase. What were the optical density or dry weight levels that defined this phase?

L 156. The bit about “Four light intensities (60, 120, 180, and 240 µmol photons...)” needs some clarification. I would like to know why they picked these intensities.

L 163. How many experimental replicates were done for each condition in this main batch?

L 223. Here, I think it is important to mention the two factors they used in the ANOVA model.

Results

L 253-254. Was there a significant difference between 180 and 240 µmol m–2 s–1?

Figure 6, upper plate. The OY-axis shows negative values, which does not make sense since removal efficiency can not be less than 0.

Discussion

L 351, 354. Change “Table 1” to “Table 4”

L 368. I think it would be a good idea for the authors to calculate and include the actual C:N:P ratio based on their COD, nitrate, and phosphate data to support this claim.

L 382-384. The authors should be a bit careful when discussing how suitable biomass from wastewater is for certain applications, taking potential contaminants into account.

Conclusion

I think this section could be shortened. The authors should focus on the main findings, their implications, and future perspectives.

Author Response

Comment 1: Introduction (L 36-39): I think it would really help if the authors could give a quick explanation or maybe an example of "qualitative degradation" besides just nutrient pollution. Adding some references here could make it clearer too.

Response 1: We agree that this term required elaboration. We have updated the Introduction (Lines 36–39) to explicitly mention specific forms of qualitative degradation, including "increased turbidity, pathogen accumulation, and the presence of emerging contaminants." We have also added two new citations [4, 5] to support this statement.

Comment 2: Introduction (L 40-42): The authors should back up their claim about the rapid growth in wastewater generation with a citation.

Response 2: We have added a relevant citation [6] regarding the rapid expansion of the food service sector and the consequent increase in wastewater generation to support this claim.

Comment 3: Introduction (L 66-67): I would suggest that the authors share the typical tolerance ranges for Spirulina in wastewater conditions.

Response 3: We have incorporated specific tolerance ranges into the Introduction. The revised text now states that Spirulina can endure "pH levels of 9.0–11.0, salinities up to 30 g L⁻¹, and free ammonia concentrations up to 100 mg L⁻¹." References [21, 22] have been added to validate these values.

Comment 4: Materials and Methods (L 117): I think it would be helpful if the authors briefly mentioned the key criteria they used to pick the optimal 75% dilution based on the screenings.

Response 4: We have clarified in Section 2.1 that the 75% dilution was selected because it supported the "highest biomass accumulation and specific growth rate" compared to other dilutions, while avoiding the toxicity observed at 100% concentration.

Comment 5: Materials and Methods (L 141): I am curious why the authors chose the stationary phase instead of the exponential phase.

Response 5: We selected stationary-phase cells for inoculation because they typically possess higher cell densities and thicker cell walls, providing greater metabolic robustness against the initial osmotic and chemical shock of the wastewater medium. We have added a brief explanation and citations [39, 40] in Section 2.3 to justify this choice.

Comment 6: Materials and Methods (L 156): The bit about light intensities... I would like to know why they picked these intensities.

Response 6: We selected the range of 60–240 µmol photons m⁻² s⁻¹ to cover the spectrum from light-limitation (<100 µmol) to saturation and potential photoinhibition (>200 µmol). This allowed us to identify the precise point where photosynthetic efficiency is maximized without inducing oxidative stress.

Comment 7: Materials and Methods (L 163): How many experimental replicates were done for each condition in this main batch?

Response 7: We have explicitly stated in Section 2.2 and Section 2.8 that all experiments, including the main batch cultivation, were conducted in triplicate (n = 3).

Comment 8: Materials and Methods (L 223): Here, I think it is important to mention the two factors they used in the ANOVA model.

Response 8: We have updated Section 2.8 (Statistical Analysis) to specify that the two fixed factors used in the two-way ANOVA were "Treatment Type" (e.g., wastewater dilution or light intensity) and "Time" (cultivation days).

Comment 9: Results (L 253-254): Was there a significant difference between 180 and 240 µmol m–2 s–1?

Response 9: No, there was no statistically significant difference. We have clarified in Section 3.2 that "No statistically significant difference (p > 0.05) was observed between 180 µmol m⁻² s⁻¹ and 240 µmol m⁻² s⁻¹." Consequently, 180 µmol was chosen as the optimal intensity to minimize energy consumption.

Comment 10: Figure 6 (now Figure 4), upper plate: The OY-axis shows negative values, which does not make sense since removal efficiency can not be less than 0.

Response 10: We thank the reviewer for noticing this error. We have corrected the y-axis scaling in Figure 4 to start at 0%, ensuring that no negative values are displayed.

Comment 11: Discussion (L 368): I think it would be a good idea for the authors to calculate and include the actual C:N:P ratio based on their data.

Response 11: This was an excellent suggestion. We used our initial characterization data (COD: 342.4 mg/L, Nitrate: 46.87 mg/L, Phosphate: 11.56 mg/L) to calculate the actual C:N:P ratio. We have added a sentence in the Discussion stating: "The wastewater exhibited a C:N:P ratio of approximately 30:4:1," and explained how this balanced stoichiometry facilitated simultaneous carbon and nitrogen assimilation.

Comment 12: Discussion (L 382-384): The authors should be a bit careful when discussing how suitable biomass from wastewater is for certain applications, taking potential contaminants into account.

Response 12: We agree that safety is paramount. We have added a cautionary statement in the Discussion emphasizing that "before any commercial application in food or feed chains, the biomass must undergo rigorous screening for potential contaminants, including heavy metals, pathogens, and cyanotoxins, to ensure safety compliance" [81].

Comment 13: Conclusion: I think this section could be shortened. The authors should focus on the main findings...

Response 13: We have condensed the Conclusion significantly. We removed general background information and focused strictly on the quantitative results (productivity, removal rates) and the direct implications for circular bioeconomy applications.